TAFII250 is one of more than 10 TATA-binding protein (TBP)-associated factors (TAFIIs) that are complexed with TBP in TFIID. Binding of TFIID to the core promoter surrounding the transcription start site of a gene nucleates assembly of the preinitiation complex (PlC), which contains RNA polymerase II (Pol II) and the general transcription factors TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH. The nucleating function of TFIID is thought to comprise several distinct events: (1) activator-dependent recognition of core promoter DNA elements; (2) the generation of a chromatin environment that is favorable to PlC assembly and transcription initiation; and (3) structural and functional modification of GTFs to facilitate PlC assembly and transcription initiation. Activities attributed to TAFII250 in vitro are consistent with roles for TAFII250 in each of these TFIID events, but evidence supporting these or other specific roles for TAFII250 in the context of chromatin is insufficient. We hypothesize that individual TAFII250 activities play gene-specific roles during RNA Pol II transcription and that signaling pathways and activators utilize individual TAFII250 activities to modulate the transcription of specific genes. Thus, in Aim 1 of the grant, we will use an unbiased genetic screen to identify TAFII250 domains and residues that are important for Drosophila viability. This screen will allow us to identify biologically relevant features of TAFII250. In Aim 2 of the grant, we will determine whether differential expression or differential chromatin association of TAFII250 C-terminal region (CTR) isoforms contributes to gene-specific transcription in Drosophila and whether TAFII250 features identified in Aim 1 contribute to this mechanism. In Aim 3 of the grant, we will determine whether differential biochemical activities of TAFII250 CTR isoforms contribute to gene-specific transcription in Drosophila and whether TAFII250 features identified in Aim 1 contribute to this mechanism. The determination of how individual TAFII250 activities contribute to the complex series of sequential interactions and conformational changes that promote transcription initiation in vivo will lead to a better understanding of mechanisms through which gene expression is regulated and how defects in this process lead to cancer and developmental abnormalities.